title: Determination of the minimum inhibitory concentration of Cryptococcus neoformans and Cryptococcus gattii against fluconazole by flow cytometry.
abstract: Recent studies have used flow cytometry (FCM) as an important alternative method to determine the antifungal susceptibility of yeasts compared to the broth microdilution Clinical and Laboratory Standards Institute (CLSI) reference procedure. We present a comparative study of the broth microdilution method and flow cytometry to assess the in vitro antifungal susceptibility of Cryptococcus neoformans (n = 16) and C. gattii (n = 24) to fluconazole. The minimum inhibitory concentration (MIC) assays by flow cytometry were defined as the lowest drug concentration that showed ∼50% of the count of acridine orange negative cells compared to that of the growth control. Categorical classification showed all C. neoformans isolates were susceptible to fluconazole. Three isolates of C. gattii were susceptible dose-dependent and the remaining 21 isolates were classified as susceptible. MICs comparison of both methodologies demonstrated 100% categorical agreement of the results obtained for C. neoformans and C. gattii. The MICs obtained with the CLSI-approved method and flow cytometry were compared by the Spearman correlation test and a significant Pv = 0.001. The flow cytometric method has the advantage of analyzing a large and constant number of cells in less time, i.e., 9 h incubation for fluconazole using acridine orange versus 72 h for broth microdilution method. In conclusion, the two methods were comparable and flow cytometry method can expedite and improve the results of in vitro susceptibility tests of C. neoformans and C. gattii against fluconazole and also allows comparative studies in vitro/in vivo more rapidly, which along with clinical data, could assist in selecting the most appropriate treatment choice.
authors.affiliation: Evandro Chagas Clinical Research Institute (IPEC), Oswaldo Cruz Foundation (FIOCRUZ), Rio de Janeiro.
authors.forename: Bernardina Penarrieta
authors.initials: BP
authors.lastname: Morales
authors.forename: Ivan Neves
authors.initials: IN
authors.lastname: Junior
authors.forename: Luciana
authors.initials: L
authors.lastname: Trilles
authors.forename: Alvaro Luiz
authors.initials: AL
authors.lastname: Bertho
authors.forename: Raquel De Vasconcellos Carvalhaes De
authors.initials: Rde V
authors.lastname: Oliveira
authors.forename: Marilia Martins
authors.initials: MM
authors.lastname: Nishikawa
authors.forename: Mônica Dos Santos
authors.initials: Mdos S
authors.lastname: Elias
authors.forename: Bodo
authors.initials: B
authors.lastname: Wanke
authors.forename: Márcia Dos Santos
authors.initials: Mdos S
authors.lastname: Lazéra
mesh_terms: D000935:Antifungal Agents / Q000494:pharmacology*; D056285:Cryptococcus gattii / Q000187:drug effects*; D003455:Cryptococcus neoformans / Q000187:drug effects*; D005434:Flow Cytometry / Q000379:methods*; D015725:Fluconazole / Q000494:pharmacology*; D008826:Microbial Sensitivity Tests / Q000379:methods; D013997:Time Factors
chemical_list: D000935:Antifungal Agents; D015725:Fluconazole
keywords: Cryptococcus; antifungal susceptibility; flow cytometry